The cluster of differentiation, also called cluster of designation (CD) is a nomenclature system used for the identification and investigation of cell surface molecules, either receptors or ligands used for immunophenotyping of cells. T cell receptors (TCR), which reside on the cell surface of T cells, can combine with CD markers forming a complex (DeJarnette et al., “Specific Requirement for CD3 in T Cell Development.” Immunology 95 (1998): 14909-4914; Dietrich et al., “Role of CD3/in T Cell Receptor Assembly.” JCB 132 (1995): 299-310). This complex can classify the type of T cell and its stage in the cell development process. CD3 is a multimeric protein complex that contains one γ, one δ, two ε and two ξ chains, which associate with TCR to form TCR complex (Dietrich et al., “Role of CD3/in T Cell Receptor Assembly.” JCB 132 (1995): 299-310). In early stages of T-cell development, CD3 is expressed in the cytoplasm and as the T cell matures, it migrates into the cell membrane (DeJarnette et al., “Specific Requirement for CD3 in T Cell Development.” Immunology 95 (1998): 14909-4914; Dietrich et al., “Role of CD3/in T Cell Receptor Assembly.” JCB 132 (1995): 299-310). CD3 is expressed in all T-cells during all stages of their development making it a specific marker for identification of T-cells (DeJarnette et al., “Specific Requirement for CD3 in T Cell Development.” Immunology 95 (1998): 14909-4914; Dietrich et al., “Role of CD3/in T Cell Receptor Assembly.” JCB 132 (1995): 299-310).
CD16 is a member of the Fc receptor family which contributes to the protective functions of the immune system and has two subtypes FcγRIIIa (CD16a) and FcγRIIIb (CD16b). Fc receptors are designed to bind antibodies that are attached to foreign cell types (Sconocchia et al., “Tumor Infiltration by FccRIII (CD16)+Myeloid Cells Is Associated with Improved Survival in Patients with Colorectal Carcinoma.” IJC 128 (2010): 2663-672). CD16 is present on the surface of natural killer cells (NK), neutrophils, macrophages, monocytes, and in rare cases activated T cells (Sconocchia et al., “Tumor Infiltration by FccRIII (CD16)+Myeloid Cells Is Associated with Improved Survival in Patients with Colorectal Carcinoma.” IJC 128 (2010): 2663-672). CD16 on NK cells can induce antibody-dependent cell-mediated cytotoxicity (ADCC) (Bojarska-Junak et al., “Natural Killer-like T CD3+/CD16+CD56+Cells in Chronic Lymphocytic Leukemia: Intracellular Cytokine Expression and Relationship with Clinical Outcome.” Oncology Reports 24.3 (2010): 803-10; Sconocchia et al., “Tumor Infiltration by FccRIII (CD16)+Myeloid Cells Is Associated with Improved Survival in Patients with Colorectal Carcinoma.” IJC 128 (2010): 2663-672; Carrega et al., “Natural Killer Cells Infiltrating Human Nonsmall-cell Lung Cancer Are Enriched in CD56bright CD16-Cells and Display an Impaired Capability to Kill Tumor Cells.” Cancer 112 (2008): 863-75). Once the Fc receptor on an NK cell recognizes foreign IgG antibody, the NK cells releases cytokines that enter the foreign cell and promotes cell death by apoptosis (Carrega et al., “Natural Killer Cells Infiltrating Human Nonsmall-cell Lung Cancer Are Enriched in CD56brightCD16-Cells and Display an Impaired Capability to Kill Tumor Cells.” Cancer 112 (2008): 863-75).
GA201, which is a glycoengineered anti-EGFR human monoclonal antibody, enhances ADCC (Gerdes et al., “GA201 (RG7160): A Novel, Humanised, Glycoengineered Anti-EGFR Antibody with Enhanced ADCC and Superior in Vivo Efficacy Compared with Cetuximab.” AACR Journals (2012): 1-32). GA201 is an IgG1 molecule whose Fc region is glycosylated to increase its binding affinity for FcγRIIIa (CD16a) on immune effector cells. Since GA201 was engineered for high ADCC effectiveness, it has demonstrated increased affinity for low and high CD16 expressed on NK cells, macrophages, and T cell sub populations (Gerdes et al., “GA201 (RG7160): A Novel, Humanised, Glycoengineered Anti-EGFR Antibody with Enhanced ADCC and Superior in Vivo Efficacy Compared with Cetuximab.” AACR Journals (2012): 1-32). This allows CD16 to be used as indicator of the level of GA201 activity in patients with tumors (Gerdes et al., “GA201 (RG7160): A Novel, Humanised, Glycoengineered Anti-EGFR Antibody with Enhanced ADCC and Superior in Vivo Efficacy Compared with Cetuximab.” AACR Journals (2012): 1-32). CD3 has also been suggested to be linked in GA201 activity through triggering of adaptive immunity. This indicates that detecting CD3 and CD16 together can be used to predict a cancer's likely response to GA201 (Bojarska-Junak et al., “Natural Killer-like T CD3+/CD16+CD56+Cells in Chronic Lymphocytic Leukemia: Intracellular Cytokine Expression and Relationship with Clinical Outcome.” Oncology Reports 24.3 (2010): 803-10).
Unfortunately reliable antibodies from different species for CD3 and CD16 are not available. This leads to undesirable cross-reactivity of secondary antibodies used to detect the CD3- and CD16-primary antibodies in a sample. Thus, methods are needed that permit detection of proteins using primary antibodies from the same species (e.g., mouse CD3 and mouse CD16 antibodies, or rabbit CD3 and rabbit CD16 antibodies).